PROJECT SUMMARY/ABSTRACT Aedes aegypti mosquitoes spread arboviruses including Zika, chikungunya, dengue, and yellow fever by biting and feeding on the blood of multiple human hosts. Understanding the mechanisms by which mosquitoes are attracted to humans and disrupting this deadly behavior will have profound implications for public health. Female Ae. aegypti utilize human cues such as carbon dioxide, heat, and odor to efficiently locate a host. These mosquitoes naturally experience periods when this strong attraction to humans is suppressed, including following a blood-meal. The mechanisms underlying host-seeking suppression are unknown. The objective of this proposal is to establish how endogenous signaling pathways regulate chemosensory circuits necessary for mosquito host-seeking behavior. The physiological state of the female mosquito changes significantly after ingestion of a blood-meal, when she must digest the protein in the blood to develop eggs. The steroid hormone 20-hydroxecdysone (20E) is critical for the egg-development process, and levels of 20E are upregulated in the hemolymph (insect blood) shortly after the blood-meal. In preliminary studies, we demonstrated that feeding 20E to non-blood-fed female mosquitoes robustly inhibited host-seeking drive. To determine if 20E is acting directly in the brain, Ae. aegypti brains were immunostained with an antibody for an isoform of the 20E receptor, ecdysone receptor isoform A (EcR-A). These studies showed that EcR-A is found in specific antennal lobe glomeruli. The antennal lobe receives projections from chemosensory neurons in the antennae, maxillary palps, and proboscis, chemosensory organs that are critical for the detection of human odor and carbon dioxide. Understanding how 20E and EcR-A function in the antennal lobe and chemosensory tissues offers mechanistic insights into the pathways involved in host-seeking behavior. In Aim 1 of this proposal we will characterize EcR-A circuitry in Ae. aegypti brains and investigate 20E-dependent changes in chemosensory neuron activity. In Aim 2 of the proposal, we will investigate how 20E and EcR-A regulate expression of olfactory receptors and genes required for chemosensory signaling machinery in the antenna and palps. It is known that the expression of olfactory receptors changes after the blood-meal, but the transcriptional mechanism for this regulation is unknown. EcR-A is a nuclear hormone receptor and transcription factor, and 20E-mediated changes in gene expression of receptors and signaling machinery in chemosensory neurons is one mechanism that can alter mosquito responses to host cues after a blood-meal. This research training plan will take place in a highly supportive laboratory environment and will develop skills in genetic manipulation, live imaging, and bioinformatics. These studies will contribute fundamental insights into olfactory processing in mosquitoes as well as provide novel targets for vector control.